Vast majority of the biomedical applications of nanotechnology focuses on the development of synthetic nanoscale entities that are produced and assembled in the laboratory to be administered to patients or applied to biological samples. However, the biological outcomes of these in vitro-assembled nanomachines are inherently transient, making them unsuitable or inconvenient for applications where sustained interventions are necessary or desirable, such as gene therapy of genetic defects or ex vivo engineering of cells for reintroduction into patients. Nanomachines that are genetically encoded and produced in living cells in which they operate would significantly advance our ability to intervene or engineer cells with sophisticated functions beyond conventional gene therapies in a sustainable fashion. However, there is a significant gap between the sophisticated nanostructures that have been designed and built in vitro, and the nanomachines that must be stable enough and able to self-assemble in living cells (particularly in mammalian cells). Our goal is to bridge this gap by designing artificial, genetically encoded nanomachines that 1) can be produced and are stable within the living cell in which they operate, 2) can sense diverse intracellular or extracellular signals, 3) can integrate multiple signals if necessary, and 4) can respond intelligently to the dynamic environment by appropriately modulating the cell's genetic and biochemical machinery. We will focus on nanomachines comprised of RNA molecules that are transcribed in the cell from DNA templates. A modular design strategy will be employed to facilitate forward engineering of complex functions from a set of available building blocks such as sensors, actuators/transducers, and biological effectors. Moreover, the molecular basis and dynamics of the RNA nanomachines will be characterized to gain insights that can be used to improve the design strategy of RNA nanomachines. PUBLIC HEALTH RELEVANCE: The goal of the proposed studies is to develop molecular nanomachines made of RNA that are produced in living cells from DNA templates. The RNA nanomachines will integrate features such as molecular sensing and gene regulation into single RNA molecules that perform complex functions in living cells. These nanomachines may become useful tools for gene therapy and basic biological studies.